The following description provides a summary of information relevant to the present disclosure and is not an admission that any of the information provided or publications referenced herein is prior art to the present disclosure.
C. difficile infection (CDI) has been on the rise worldwide over the last several years. The clinical and economic consequences are substantial, with more than half a million cases and estimated costs of 3.2 billion dollars per year for CDI management in the U.S. alone (O'Brien, J. A., et al., Infect. Control Hosp. Epidemiol., 2007. 28(11): p. 1219-27).
CDI is an inflammatory condition of the large bowel characterized by diarrhea and can range in severity from mild to fulminant. More severe CDI syndromes are pseudomembraneous colitis and toxic megacolon. Most CDI cases occur in elderly patients in a hospital setting or in nursing homes. Hospitalization, however, increases the risk of colonization also for healthy adults. In the U.S., CDI hospitalizations and CDI-related case-fatality rates doubled between 2000 and 2005. A number of recent outbreaks have been reported in which CDI cases were primarily clonal in nature. A strain type classified as BI/NAP1/027 was responsible for more than half of the cases, and hallmarks of this epidemic “outbreak” strain are high morbidity and mortality, higher resistance to antibiotics (e.g. fluoroquinolones), the presence of a tcdC variant gene, and toxin hyper-production (Freeman, J., et al., Clin. Microbiol. Rev., 2010. 23(3): p. 529-49; Rupnik, M., M. H. Wilcox, and D. N. Gerding, Nat. Rev. Microbiol., 2009. 7(7): p. 526-36).
Use of antibiotics is a strong predisposing factor for CDI due to the disruption of the normal gut flora that otherwise suppresses C. difficile. Ingestion of spores is the main route of colonization of the human gut by C. difficile. Spores are extremely resistant to disinfectants and can persist in the environment for more than 12 months with little loss of viability or pathogenicity. Spores are also implicated in the 20-25% of CDI cases which relapse after treatment. Current treatment regimens for CDI are vancomycin or metronidazole. Several new, more selective agents that hold promise to reduce CDI recurrence rates are in clinical development.
The inflammation of the intestinal lining is caused by two toxins (toxin A and toxin B) that are expressed by some C. difficile strains. Toxin A and toxin B are glucosyltransferases that target small host GTPases in the Ras superfamily. They are encoded on the 19.6 kb pathogenicity locus and strains lacking these toxin genes are non-pathogenic. Toxinogenic strains can be further classified into toxinotypes according to sequence variability within the pathogenicity locus. Both toxins contribute to CDI, as shown by using isogenic mutants that produced either toxin A or toxin B alone and were cytotoxic in vitro and virulent in vivo (Kuehne, S. A., et al., Nature, 2010. 467(7316): p. 711-3). A vaccine prototype based on inactivated toxins A and B (toxoids) and anti-toxin monoclonal antibodies are being studied for their effectiveness in preventing recurrent CDI.
Toxin A and toxin B are structurally related, large toxins of MW˜300 kDa, and consist of an amino-terminal catalytic domain (glucosyltransferase), a central peptidase C80 domain, a translocation domain, and multiple carboxy-terminal β-hairpin repeats. The mechanism of action of the clostridial toxins has been shown to involve binding of these β-hairpin repeats to carbohydrates present on the surface of gastrointestinal cells, endopeptidase-mediated cleavage, and internalization of the catalytic domain (Pfeifer, G., et al., J. Biol. Chem., 2003. 278(45): p. 44535-41).
Some C. difficile strains produce a binary toxin which possesses ADP-ribosyltransferase activity. Although its role in pathogenesis is unclear, the presence of binary toxin is a good marker for the epidemic outbreak strain BI/NAP1/027. The binary toxin consists of two subunits, which are the actin ADP-ribosyltransferase binary toxin A chain and the pore-forming binary toxin B chain. They are secreted from the bacterial cells as separate polypeptides and have the potential to combine to form a potent cytotoxin which has been shown to kill Vero cells (Sundriyal, A., et al., Protein Expr. Purif., 2010. 74(1): p. 42-8).
Rapid and accurate CDI diagnosis is important for patient care, infection control and surveillance. The C. difficile toxins A and B are of high clinical diagnostic relevance since they are sufficiently pathogen-specific targets and the demonstration of their presence is important for CDI diagnosis. All currently used CDI diagnostic tests are qualitative and belong to one of three types, (i) cytotoxin assay (tissue culture), (ii) non-molecular toxin tests (EIA), and (iii) molecular tests (PCR).
The tissue culture-based cytotoxin assay is considered the gold standard, but is cumbersome and not routinely performed by most clinical laboratories. In essence, this assay detects C. difficile toxin via the toxin's cytopathic effect in cell culture that can be neutralized with specific anti-sera. The cytotoxicity assay detects as little as 10 pg of toxin B and is the recommended confirmatory test for 510(k) submissions in the “Draft Guidance for Industry and Food and Drug Administration Staff Establishing the Performance Characteristics of In Vitro Diagnostic Devices for the Detection of Clostridium difficile” that was released in November 2010 FDA, http://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/GuidanceDocuments/uc m234868.htm.2010.
Molecular tests for CDI are available from several diagnostic companies. The Cepheid GeneXpert™ test is based on multiplex PCR (tcdB, cdt, tcdC), with advertised sensitivity and specificity of >95% and time-to-result of 30 min. The Meridian Illumigene™ C. difficile test detects the presence of the toxin producing region by isothermal loop amplification and advertised to provide results in under an hour. The BD GeneOhm™ C diff assay is a real-time PCR method for the detection of toxin B gene (tcdB) direct from stool samples, with an assay protocol time of less than two hours, sensitivity of 93.8% and specificity of 95.5%. Gen-Probe offers the Prodesse ProGastro Cd test which also detects the toxin B gene (tcdB) by PCR and is advertised to provide results in three hours with a sensitivity of 91.7% and specificity of 94.7%.
Non-molecular tests for C. difficile toxin detection in stool samples from patients with suspected CDI are also available. Enzyme immunoassays (EIAs) are the most widely used rapid detection methods for C. difficile common antigen and toxin A/B antigens, but traditional EIAs have modest sensitivity and specificity. Among the well-type EIAs, the Meridian Premier™ Toxins A/B test and the Techlab TOX A/B II™ test are considered the best-performing ELISAs and detect both toxins in stool specimens in less than 1 hour. These assays had about 80% sensitivity and 98% specificity when tested independently. The toxin B antibodies for the Premier™ Toxins A/B (Meridian) and for the C. difficile TOX A/B II™ (TechLab) were able to detect 125 pg and 250 pg of toxin B, respectively, when tested side by side (Novak-Weekley, S. M. and M. H. Hollingsworth. Clin Vaccine Immunol, 2008. 15(3): p. 575-8). Many other well-type EIAs assays have been brought to market (GA's C. difficile antigen, R-Biopharm's Ridascreen™ Toxin A/B; Remel's ProSpect™ Toxin A/B) but are used less often in the U.S. Membrane EIA assays performed with lateral flow devices are the Meridian ImmunoCard™ Toxins A&B, the Techlab Tox A/B Quik Chek™, and the Remel Xpect™ assays.
There is one automated test on the market, bioMérieux's VIDAS™ C. difficile Toxin A&B, which combines toxin testing and culture based identification with the API® 20A strip and automated bacterial genotyping with the DiversiLab® system.
Aptamer-based C. difficile toxin tests, like EIAs, have the advantage over molecular tests that they do not require big investments in equipment or expensive reagents. Aptamers have several distinct advantages over antibodies that are currently used in non-molecular assays, such as EIAs: aptamers generally have lower molecular weight, provide higher multiplexing capabilities (low cross-reactivity, universally-applicable assay conditions), chemical stability (to heat, drying, and solvents, reversible renaturation), provide ease of reagent manufacturing, consistent lot-to-lot performance and can be produced at lower cost.
Aptamers can be generated against virtually any protein target, not only toxins A/B, but also binary toxin for which there is no antibody-based test of which Applicants are aware. Detection and read-out methods can be the same as for existing tests, thus minimizing equipment needs and training requirements.